Systematic Review of Zinc’s Benefits and Biological Effects on Oral Health

Background: This review was based on the following question: “What is the state-of-the-art regarding the effect of zinc exposure in the oral cavity on a population of adults and children, compared to dental products containing materials other than zinc, considering in vivo (clinical trials and observational studies) and in vitro studies?” according to a PICOS strategy format. This study aims to analyze zinc application in dental materials, with different compositions and chemical formulations, considering how mechanical and biological properties may influence its clinical applicability. Methods: In vivo (clinical trials: controlled clinical trials (CCTs) and randomized controlled trials (RCTs); and observational studies: case control and cohort studies) trials or in vitro studies published in English or Italian during the last 10 years on children and adult patients with zinc exposure were included by three different reviewers using the MEDLINE (via PubMed), Scopus, and Web of Science electronic databases. Results: Titles and abstracts were evaluated following the eligibility criteria. The full texts of eligible studies were then reviewed against the inclusion/exclusion criteria. Scientific and technical information of the 33 included studies were collected into evidence tables, reporting data on in vivo and in vitro studies. A narrative approach was adopted. Conclusions: Antibacterial activity was found to be the most studied property of zinc, but further investigations are needed to establish adjuvant zinc therapies in patients with oral disease.


Introduction
Zinc (Zn) is a very important trace element, and maintaining its optimal level in the human body is very important for healthy growth and development [1,2].In the human body, zinc is found in muscles (60%), bones (30%), and skin (5%) [3].Its functions include its involvement in the activation of various enzymes and proteins [4]; it also contributes to the absorption of vitamin A, vitamin E, and folate [2].Low zinc levels may be associated with an increased chance of developing infections and degenerative diseases [2].As with other micronutrients that fall into the mineral category, the amount of zinc needed varies from 2 to 13 mg/day depending on the life stage and sex of the individual, with the upper limit of zinc set at 40 mg/day [5][6][7].
In the oral cavity, it is naturally present at various sites such as saliva, dental plaque, and dental hard tissues such as the hydroxyapatite of dental enamel [5].The concentration of zinc in the enamel surface varies between 430 and 2100 parts per million (ppm), and its deposition occurs mainly before tooth eruption [6].Zinc is also important for maintaining periodontal health because of its local and immunological effect on the soft tissues of the oral cavity.It contributes to the formation of healthy teeth [5] and is used in mouthwashes and toothpastes for its important role in preventing plaque and tartar formation [3] and reducing bad breath [3].
Zinc has been involved in the composition of dental biomaterials and orthodontic materials due to its immune properties and its effects on cell division and skeletal development [8].Clinical studies have also shown that zinc ions reduce the rate of enamel demineralization [3].It has been widely demonstrated to be effective against common prevalent oral health problems, such as caries, gingivitis, and periodontitis.Due to its antibacterial and anti-matrix metalloproteinase activity, zinc has been incorporated into experimental composite resins [9], glass ionomer cements [10], adhesive resins [11,12], and desensitizing solutions [13], the latter being particularly useful for the treatment of root dentin demineralization [14].
In recent decades, many researchers have also reported the antibacterial activity of zinc oxide nanoparticles (ZnONP) [15] on various microorganisms, such as Escherichia coli [16], Campylobacter jejuni [17], Pseudomonas aeruginosa [18], and Vibrio cholerae [19].Currently, modern nanotechnology is moving toward the study of zinc-based materials to produce, for example, light-curing restorative composite resins and related adhesive systems, impression materials, dental implants with surfaces coated in zinc, mouthwashes, and root canal fillings with an antibacterial effect [20,21].
However, although positive effects of new zinc-containing dental materials have been shown in the literature, in order to completely replace the traditionally used materials, in vivo studies would be needed to confirm the properties under real conditions and with a larger sample of patients.In addition, the potential toxicity of nanomaterials has attracted the attention of researchers, but there are no recent studies on this issue [22,23].
The purpose of this review is to investigate the state of the art with respect to the application of zinc in dental materials in different dental fields and with different compositions and chemical formulations, considering how mechanical and biological properties may influence its clinical applicability.
Null hypothesis: the addition of zinc to dental materials affected biological and mechanical properties.

Materials and Methods
This review was conducted based on the following question: "What is the state-of-theart regarding the effect of zinc exposure in the oral cavity on a population of adults and children, compared to dental products containing materials other than zinc, considering in vivo (clinical trials and observational studies) and in vitro studies?"This question was created according to the PICOS strategy format [24] (Table 1) and was not registered online.The present study will include studies published in English or Italian published in the last 10 years, including in vivo trials (clinical trials: controlled clinical trials (CCTs) and randomized controlled trials (RCTs); and observational studies: case control and cohort studies) and in vitro studies on children and adult patients with zinc exposure as a variable and no zinc exposure as comparator/control.
Exclusion criteria: systematic reviews, animal studies, case reports, review articles, editorials, opinions, surveys, guidelines, conferences, commentary articles, studies on other chemical compounds, studies with no full-text available.
Primary outcome: Oral health.
The search process was performed by three different reviewers using the MEDLINE (via PubMed), Scopus, and Web of Science electronic databases.The search strategy was outlined based on PubMed MeSH terms, as shown in Table 2, and adapted for the other databases.This review was conducted based on the PRISMA statement and checklist [25] (Supplementary Table S2, available online).The screening process was performed on the titles, abstracts, and full-texts of the articles that potentially met the inclusion criteria.In each phase, three reviewers independently assessed each article.Any disagreement was resolved through discussion or by consulting other authors.The PRISMA flow diagram (Figure 1) was used to report the included and excluded studies.The electronic search was followed by a manual search of the references list of included articles.
Three independent reviewers evaluated the titles of the articles initially retrieved in the search following the eligibility criteria, and those with no relevance were excluded.Articles compatible with the inclusion criteria were selected for further examination and the abstracts were screened.The full texts of potentially eligible studies were then independently reviewed against the inclusion/exclusion criteria by the reviewers, and any disagreement was resolved by consultation with the other authors.Scientific and technical information were collected into two evidence tables (Tables 3 and 4) with Microsoft Office Excel, reporting data about in vivo and in vitro studies, respectively, with Table 3 including the following: author(s) and year of publication, population characteristics, zinc application and chemical composition, control group, biological properties and effects on oral health, principal findings, fundings, and quality assessment score; and Table 4 including the following: author(s) and year of publication, fundings, cell types or microbial strains, zinc application and chemical composition, specimen characteristics, control group, biological properties and effects on oral health, principal findings, and quality assessment score.For data analysis, a narrative approach was adopted.Three independent reviewers evaluated the titles of the articles initially retrieved in the search following the eligibility criteria, and those with no relevance were excluded.Articles compatible with the inclusion criteria were selected for further examination and the abstracts were screened.The full texts of potentially eligible studies were then independently reviewed against the inclusion/exclusion criteria by the reviewers, and any disagreement was resolved by consultation with the other authors.Scientific and technical information were collected into two evidence tables (Tables 3 and 4) with Microsoft Office Excel, reporting data about in vivo and in vitro studies, respectively, with Table 3 including the following: author(s) and year of publication, population characteristics, zinc application and chemical composition, control group, biological properties and effects on oral health, principal findings, fundings, and quality assessment score; and Table 4 including the following: author(s) and year of publication, fundings, cell types or microbial strains, zinc application and chemical composition, specimen characteristics, control group, biological properties and effects on oral health, principal findings, and quality assessment score.For data analysis, a narrative approach was adopted.
The Quality Assessment for Diverse Studies (QuADs) tool [26] (Supplementary Figure S1, available online) was selected and performed by two independent reviewers for each included study to assess the inherent quality, assigning a reliability score.The tool consists of a set of 13 items with a score from 0 (incomplete information) to 3 points (complete information).

Results
The flow diagram (Figure 1) shows a total of 442 papers after the electronic search, with 392 potentially eligible articles in English.A total of 261 studies were excluded after title screening.Then, abstract screening was completed on 131 studies, and 62 papers were eligible for full-text review.Finally, 33 studies were included for data extraction.Two The Quality Assessment for Diverse Studies (QuADs) tool [26] (Supplementary Figure S1, available online) was selected and performed by two independent reviewers for each included study to assess the inherent quality, assigning a reliability score.The tool consists of a set of 13 items with a score from 0 (incomplete information) to 3 points (complete information).

In Vivo Studies
As reported in Table 3, zinc mechanical properties, like restorations adhesive strength or flexural strength, were not considered in any of the five included studies.Regarding the biological properties evaluated, three studies analyzed the microbiological properties and effects of many bacterial species of different plaque samples using toothpaste formulations with zinc [29, 37,41]; and two studies evaluated antibacterial effects considering endodontic tissue repair and lesion sterilization after treatments with zinc oxide eugenol paste (ZOE) [48] and zinc oxide-ozonated oil [27].The clinical and radiographic effectiveness of pulpectomy agents were also taken into consideration [27,48].Prasad et al. [29] observed that toothpastes with 0.96% zinc (zinc oxide, zinc citrate), 1.5% L-arginine, and either 1450 ppm or 1000 ppm of fluoride caused significant reductions in oral bacteria compared to fluoride alone.Sreenivasan et al. [41] confirmed these findings, while Hagenfeld et al. reported no significant difference in zinc-substituted carbonated hydroxyapatite dentifrice compared to toothpaste containing an amine fluoride/stannous fluoride [37].Doneria et al. highlighted how zinc oxide-ozonated oil had 100% endodontic clinical success and was comparable to Vitapex formulation at 6 and 12 months [27].Moura et al. also reported the same clinical and radiological efficacy of zinc oxide eugenol (ZOE) paste compared to a formulation of chloramphenicol, tetracycline, and zinc oxide [48].

In Vitro Studies
Information about the in vitro studies is reported in Table 4, considering applications in various dental fields: conservative and cariology, endodontics, periodontology and implantology, orthodontics, and other aspects.Microbiologic analysis (CFU) on anaerobic organisms, Gram-negative bacteria and malodor bacteria of dental plaque, tongue scrapings, and cheek surfaces.
Significant reductions in functional bacterial groups from distinct oral niches compared to control group (p < 0.05).Reductions between 42 and 68% for anaerobic bacteria 12 h after brushing, increasing to 46-80% 4 h after brushing; and between 49 and 61% for Gram-negative bacteria, that increased to 54-69% 4 h post-brushing.Significantly reduced amount of S. Mutans (p < 0.05); DC was enhanced; ion release analysis revealed stability of Zn 2+ (as in the 5 wt.% group); even after 9 cycles of a 24 h wash.Compressive strength was significantly reduced (p < 0.05) just in the 5% ZDMA group, while the other groups were superior in comparison to the control.For the dentine shear bond strength, only the 5% ZDMA group was significantly higher than the control (p = 0.000).Zinc ions' effect on the growth of oral bacteria was strain-dependent.F. nucleatum was the most sensitive and suppressed by media containing 0.001% zinc ions.There was an inhibitory effect on oral malodor with direct binding with gaseous H 2 S and suppressing the growth of VSC-producing oral bacteria.The scaffolds seemed to support the adhesion and proliferation of human dental pulp stem cells and were hemocompatible with human red blood corpuscles.The scaffolds were found to be antibacterial and mildly antibiofilm against S. aureus.

None
Antibacterial effects and metabolic activity on fibroblasts and endothelial cells, and biocompatibility with chicken chorioallantoic membrane assay (CAM)l flow cytometry for cell death; cell viability assay for cellular metabolic activity (CMA) with the alamarBlue™ Cell Viability Reagent.
ZnO NPs had favorable properties for biomaterials modification and could help to guide the tissue reaction and promote complication-free healing (p ≤ 0.001).

Conservative and Cariology
Two studies applied Zn in dental adhesive containing zinc dimethacrylate ionomer (ZDMA) [38] and ZnO [50], one study used an Ag/ZnO nanocomposite [42], and one study used elastomeric temporary resin-based filling materials with zinc methacrylate [32].Barma et al. used a ZnO-NP varnish [49], Lavaee et al. used zinc sulfate and zinc acetate [30], and Suzuki et al. used metal chlorides (ZnCl 2 ) and metal acetates ((CH 3 COO) 2 Zn) [33]; while one study used solutions prepared with nano ZnO powder [39], one study used solutions prepared with zinc-doped nanoparticles (Zn-NPs) [13], one study used solutions prepared with divalent cation Zn 2+ alone and in combination with ZnCl [43], and one study used solutions prepared with zinc-substituted hydroxyapatite/alendronate-grafted polyacrylic acid hybrid nanoneedles [44].Manus et al. applied a dentifrice with zinc citrate formulations with increasing replacements of zinc oxide (a water-insoluble source of zinc ions) to generate a dual-zinc active system [31], and Tabatabaei et al. applied zinc toothpastes and mouthwashes [40].

Antibacterial Properties
Five articles evaluated the antibacterial effects on S. mutans: growth [38,49], biofilm, acid production, and antioxidant potential [49]; biofilm formation, virulence factors, and related genes expressions at sub-minimum inhibitory concentrations (MIC) [42]; inhibitory and bactericidal effects, MIC, and minimum bactericidal concentration (MBC) [30]; and biofilm formations and growth and EPS extraction [43].Eskandarizadeh et al. reported an excellent antimicrobial property against S. mutans [38].According to Barma et al. [49], a significantly suppressed expression of S. mutans' virulence-factors-related genes was observed [42].Zinc sulfate and zinc acetate salts had an inhibitory effect on S. mutans, and zinc sulfate solution caused higher MIC and MBC compared to penicillin and chlorhexidine [30], while Steiger et al. reported strong effects but no significance [43].
One article studied bactericidal properties via a biofilm model after the formation of an acquired pellicle, considering metabolic activity and live/dead staining, highlighting how ZnO adhesive caused lower CFU/mL not only in S. mutans but also in other microorganisms with lower metabolic activity and higher dead bacteria [50].One article evaluated growth, MIC, and MBC on S. mutans, E. faecalis, L. fermentum, and C. albicans, observing the greatest antimicrobial effect against S. mutans and E. faecalis [39].In another article, the antimicrobial effects on E. faecalis and a S. mutans were studied [32].The inhibitory effects on bacterial growth were also analyzed according to Zn ions' binding to gaseous hydrogen sulfide (H 2 S) and their minimum concentration required to inhibit H 2 S volatilization, and Suzuki et al. showed how zinc ions' effect on the growth of oral bacteria was strain-dependent (F.nucleatum was the most sensitive) [33].There was also an inhibitory effect on oral malodor via direct binding with gaseous H 2 S and a volatile sulfur compound-producing oral bacteria suppression [33].Zn bioavailability enhancement, penetration and retention, and antibacterial efficacy were also considered, and Manus et al. highlighted a significant bacterial metabolic activity reduction related to bacterial glycolytic function and total oxygen consumption [31].

Physical, Chemical, and Mechanical Properties
The physical, chemical, and mechanical properties analyzed were degree of conversion (DC) in three studies [32,38,50]; Zn ion release amount, compressive strength, and shear bond strength in only one study, which observed that compressive strength was significantly reduced and dentine shear bond strength significantly higher for 5% ZDMA [38]; flexural strength and elastic modulus in only one paper, in which ZnO incorporated at 7.5% caused lower flexural strength, while at 2.5%, it had a greater modulus of elasticity [50]; and microleakage, water sorption/solubility, depth of cure, ultimate tensile strength, and Shore D scale hardness in one study, which reported lower microleakage and water sorption and higher ultimate tensile strength values [32].
Zinc showed strong antibacterial properties in all the formulations used: in combination with Ag [34] or incorporated into endodontic cements [28] or adhesives such as zinc oxide quantum dots (ZnOQDs) [12] or zinc chloride (ZnCl 2 ) [45].In particular, the combination with silver revealed a powerful effect against planktonic and biofilm-resident E. faecalis related to the depolarization of the bacterial membrane with the addition of Zn 2+ .Garcia et al. also reported an antibacterial activity against biofilm formation and planktonic bacteria, which did not affect the adhesives' physicochemical and mechanical properties [45].Furthermore, no differences, in terms of biological and physical properties, were reported when comparing zinc oxide eugenol cements to other endodontic cements (MTA) [28].
Zinc improved antibacterial properties when used as a coating metal [52], promoted an inhibitory effect on Gram-negative anaerobes and on A. actinomycetemcomitans [46], and suppressed S. aureus biofilm formation [51], but reductions in other bacterial viabilities were more pronounced with other metal nanoparticles [35].While Vergara-Llanos et al. [53] highlighted how zinc antibacterial activity in a monospecies model was strain-dependent and induced mitochondrial dose-dependent cytotoxicity, increasing LDH release and intracellular ROS generation [53], zinc also promoted initial cellular adhesion, proliferation, and osteogenic differentiation [51], the critical force of scratching and corrosion resistance in association with cytocompatibility [52], and good roughness properties [35].

Orthodontics
Three articles analyzed the properties of zinc related to orthodontics aspects [36,54,57].These articles evaluated zinc properties considering its application on composite resin with silver/zinc oxide nanoparticles [54], bonding agents containing zinc-doped bioactive glass [36], and brackets coated with nanoparticles of ZnO or Ag/ZnO [57].The biologi-cal properties studied were ion release, cytotoxicity, biocompatibility, and antimicrobial properties [36,54], as well as antibacterial activity on S. mutans and L. acidophilus [57].
Zn exhibited adequate degradation behavior and cell compatibility and favorable antibacterial properties in the oral environment [55].Zinc scaffolds were compatible to human red blood corpuscles and had mildly antibacterial and antibiofilm effects against S. aureus [47].Furthermore, ZnO nanoparticles showed good properties for biomaterials modification and could promote tissue reaction and complication-free healing [56].
Table 5 summarizes the characteristics of the evaluated zinc dental products and their main effects.

Discussion
The results highlighted in the literature and the data obtained in this review show that zinc is indispensable for oral health and therefore an element found in many dental materials, not only those destined for home hygiene.In fact, zinc is increasingly being used to make modern, affordable, and valuable materials for the prevention of oral diseases.Zinc is effective in the prevention and treatment of aphthous ulcers, dental caries [58,59], and periodontal disease [60].It promotes remineralization and inhibits the dissolution of dental hard tissues.It has also been identified as a biomarker of oral squamous cell carcinoma and should therefore be evaluated for suspicious lesions [58].It would therefore be crucial to increase knowledge of the role of this element and the outcomes of its deficiency, considering the naturally available food sources for a healthy and complete diet and how other elements compete for its absorption, such as iron supplements.In vulnerable individuals, such as the immunocompromised, patients undergoing chemo-or radio-therapy, patients with oral disease, pregnant women, and children, it is therefore extremely important.Certainly, zinc deficiency could lead to the onset of oral pathologies; however, since there are very few evaluable studies in the literature [1], before a clinical protocol of zinc's supplementation as a co-adjuvant therapy to specific treatments can be established, further in vivo research with adequate patient sample sizes and long-term follow-up is needed.
Thus, this review aims to provide an overview of the properties and characteristics of zinc in various dental fields and its biological effects in the oral cavity, examining studies on healthy and unhealthy patients or on pathological or non-pathological cell populations.
The null hypothesis was rejected; the addition of zinc to dental materials did not affect biological and mechanical properties.
In this review, we included only five in vivo studies.Interestingly, all authors focused on the antibacterial power of zinc, albeit using different materials and therefore being difficult to compare.It is obviously evident how important it is to minimize the number of pathogenic bacteria present inside the oral cavity during daily hygiene practices using the mechanical action of brushing combined with the chemical action of zinc, but how clinically important bacterial control is during endodontic therapies it is also evident given the impossibility of achieving complete sterility of the endo canal system, especially in association with periapical lesions.It would be interesting to investigate this topic with further studies by increasing the numerosity and homogeneity of the sample under consideration, and also taking into account the adult population since, with regard to the use of zinc in endodontic medications, only pediatric subjects have been considered, and there are no similar studies analyzing the same properties in adult patients at present.
In vitro evaluation of materials used in conservative therapy and endodontics containing zinc also showed that the main property examined by the authors was zinc's antibacterial capacity.Zinc demonstrated excellent effects in all the formulations examined, although their therapeutic applications and clinical modalities of use are so different from each other that it is a complex matter to perform an accurate comparison.The wide variability in the studies is also reflected in what is clinically expected from the reconstructive or three-dimensional endodontic medication and obturation materials, respectively, although the possibility of having an effective antibacterial effect is certainly the common element in both.The variability in the different types of tests used to analyze the properties under investigation is also a direct consequence of the heterogeneity of the studies.The addition of zinc does not seem to affect the activity of the materials, and there is no evidence of negative mechanical performance [45]; indeed, in some cases, an improvement has been shown, with lower microleakage and water sorption and higher ultimate tensile strength [32]; however, this is an aspect to be given more consideration in future studies as high mechanical performance is required for such materials, especially when used in pedodontics, and it would be crucial to combine a good antibacterial effect with improved mechanical features.
Nanotechnology used in conservative dentistry is an integral part of the latest dental research.ZnO NPs are biocompatible and possess antimicrobial action against a wide range of microorganisms.The antibacterial properties appear to increase with increasing particle surface area, but issues regarding toxicity and particle release patterns, as well as long-term properties, still need to be resolved.Unfortunately, particle dispersion could increase flexural strength, decrease shear strength, and decrease compressive strength in composite materials [61,62].
In contrast, the application of zinc in periodontology has led to results with obvious discordance, even considering the antibacterial aspect.Materials with heterogeneous chemical compositions and different clinical uses have certainly been used, but all authors have evaluated the use of zinc as a coating material or for implant/prosthetic use.In fact, in some studies, zinc has been observed to have improved antibacterial properties [46,51,52], while Sánchez et al. have found that the reduction in bacterial viability was more pronounced with the use of other types of metal nanoparticles [35].Other recent reviews also agree on the antibacterial properties of zinc [63,64].Zn used in various products has been shown to be effective against common prevalent oral health problems, such as dental caries, gingivitis, and periodontitis, as it exhibits good oral substantivity and is retained in plaque and saliva for many hours; also, with repeated applications, there is a build-up effect in plaque [3].Almoudi et al. [63] reported significant efficacy in terms of the inhibition of S. mutans growth by zinc, even at low concentrations, indicating that it can be used safely in oral products.However, Zn has low enamel remineralization potential, and no net effect on caries has been reported with in vitro studies [3].Griauzdyte et al. [64] supported the antibacterial action of zinc against periodontal pathogenic bacteria, confirmed by Liu et al. [65], suggesting that zinc may play a key role in immune defense, inflammatory responses, and bone remodeling, and that its homeostasis is essential for periodontal regeneration.The integration of zinc within an appropriate dosage range or the regulation of zinc transport proteins could potentially enhance regeneration by boosting immune defenses or promoting cell proliferation and differentiation [65].
Regarding cytotoxicity, Vergara-Llanos et al. [53] pointed out how zinc induces dosedependent mitochondrial cell toxicity, while Lin et al. noted how zinc is cytocompatible [52].Furthermore, denture adhesives with zinc could be responsible for decreased cell viability, ROS production, aberrant cell morphologies, and the induction of apoptosis and cell death [66].Zn cytotoxicity was also found in ZOE-based dental materials during the initial setting phase with immortalized human dental pulp stem cells [67] and three-dimensional (3D) cultures of immortalized human oral keratinocytes [68]; such moderate or severe cytotoxic reactions appear to be related to the constant dissolution of such materials when exposed to an aqueous environment for prolonged periods.Another study suggested that in a model of non-keratinized oral epithelial cells at concentrations exceeding 0.031% w/v, ZnO was the most cytotoxic nanomaterial among several common oral hygiene products because nano-size effects have some impact on the cytotoxicity of a material [69].Significant toxic effects of ZnO-NP were also found at concentrations of ~50-100 µg/mL on human periodontal ligament fibroblasts, depending on the concentration and duration of exposure [70].Although it should be noted that the authors used different types of assays, materials, and cells, it would be interesting to understand whether it is zinc alone that causes cytotoxicity or whether it is because it is incorporated into other dental materials.The same problems also occurred from the orthodontic point of view since the chemical formulations of the materials studied and their use were very heterogeneous, as were the biological and mechanical properties and the tests used for their evaluation; as a result, it is extremely difficult to make realistic comparisons between the various studies.

Limitations of the Study
QuADs allows for the qualitative analysis of selected papers with reliability, but there are some limitations to be taken into consideration.We extended the investigation of zinc's effects to both in vivo and in vitro studies, including both healthy human or pathological cell populations, to obtain a complete overview of zinc's biological and mechanical effects.Comparison of these studies, although reliable, is difficult; in the case of the in vivo studies, this difficulty can be due to the enrollment of a minimal sample size or non-homogeneous patient populations, especially with regard to age.In addition, the chemical compositions of the materials used are extremely variable, both in terms of the types of materials and the percentages of zinc used, either isolated or bonded to other components, which certainly influence the overall effect.It will also be important to carry out further research taking into consideration some of the important limitations found in this study, such as the inclusion of work in languages other than English and Italian and the use of new materials in innovative fields of dentistry dedicated to patients with special needs or to fragile or geriatric patients.In the literature, there are still few in vivo studies, and there are no studies on 3D tissue or organoid models.It is important to obtain more information regarding zinc in in vivo conditions in order to precisely define its therapeutic use in various formulations.

Conclusions
It is clear that the property of greatest interest in the use of zinc is its capacity to achieve an antibacterial effect; however, further investigation is needed in order to establish precise adjuvant zinc therapies in patients with oral disease.
In consideration of the results obtained and the limited in vivo material in the literature on this subject, from this review comes the intention of the research group to set up a collateral in vivo study to analyze the efficacy of plaque removal by establish a comparison between toothpaste containing zinc and fluoride and standard fluoride toothpaste.

Materials 2024 , 32 Figure 1 .
Figure 1.Flow diagram of the screening and selection process according to the PRISMA statement.

Figure 1 .
Figure 1.Flow diagram of the screening and selection process according to the PRISMA statement.

Table 1 .
Research question with the PICOS strategy format and inclusion/exclusion criteria.
Studies on other chemical compounds or with no full-text available, systematic reviews, animal studies, case reports, editorials, opinions, surveys, guidelines, conferences, commentaries

Table 3 .
Characteristics of in vivo studies included.

Table 4 .
Characteristics of in vitro studies included.µg/mL and 1.3 µg/mL, respectively.A total of 0.1 mg/µL had the greatest zone of inhibition (24 mm).A total of 0.1 mg/µL inhibited 90% of S. mutans biofilms and exhibited antioxidant capacity in a dose-dependent manner (94% inhibition, 100 µg/mL).A total of 0.1 mg/µL ZnO-NP caused very low cytotoxicity to Hep G2 cells and was non-cytotoxic to HEK-293T cells.

Table 5 .
Types of products used with zinc and their properties.